2.1. In Vitro Germination of Pollen
Pollen in flowering plants is generally produced in staminate flowers. In corn, for example, such staminate flowers occur in clusters (the tassel) at the top of the plants (reviewed in Goss, 1968, Bot. Rev. 34:333-358). Three stamens are formed within each flower with elongated anthers that open at the tips following anthesis. During meiosis, microspores are produced in the anthers which subsequently develop into pollen grains. Such pollen grains are released and dispersed by the wind. As a result, self-pollination, and/or cross-pollination may occur. During pollination, the pollen grains are caught on stigmas of the pistillate flowers which are borne in clusters lower on the plant which make up corn silk and begin to germinate within one to one and a half hours. Pollen tubes are usually well established in the corn silks two hours after pollination.
Pollen produced, however, must be viable for a long enough period of time for pollination to occur. Therefore, studies of pollen viability are of great practical as well as theoretical value. Environmental conditions may adversely affect pollen viability and, ultimately, fertilization due to the transportation of pollen for breeding purposes over long distances. Furthermore, pollen is not always produced at the time or place where needed for plant breeding studies.
During pollen grain development, the maize microspore divides to form the generative and tube nuclei. The generative nucleus divides again to produce two crescent-shaped sperm cells. Thus, the pollen grain of maize has three nuclei (trinucleate) when released from the anther. Brewbaker and Majumbder (1959, Adv. Bot. 1:1503-1508) showed that whereas binucleate pollen grains germinate very well. Other agronomic crops with trinucleate pollen include Brassica (vegetable and oilseed rape) and Triticum (wheat).
Numerous studies have focused on the in vitro germination of pollen grains in a number of crops. Several factors may influence the in vitro germination of such a crop. These include the particular genotype which is being cultivated, how long the plant was stored after anthesis before obtaining the pollen, and the composition of the media. Procedures used in the art generally involve incubating the pollen grains on a solid substrate (e.g. agar) containing a given culture medium until there is a profusion of pollen tubes from germination pores (reviewed in Goss, 1968, Bot. Rev. 34:333-358). There is a general consensus that calcium, boron, and an osmoticum are critical in obtaining pollen germination in a variety of flowering plants, examples which include corn (Cook and Walden, 1967, Can. J. Bot. 45:605-613; Pfahler, 1968, Can. J. Bot. 46:235-240; and Goss, 1968, Bot. Rev. 34:333-358), Brassica (Roberts et al., 1983, Theor. Appl. Genet. 65:231-238), and petunia (Brewbacker et al. 1963, Am. J. Bot. 56:861-865). The osmoticum generally used is a sugar. (Goss, 1968, supra and Portnoi et al., 1977, Ann. Bot. 41:21-27). The most frequently used sugar when germinating pollen in vitro is sucrose.
The effects of other substances on in vitro germination of pollen has also been tested. For example, polyethylene glycol-400 (PEG-400) has been proposed as a substitute for sugar for germinating Petunia pollen since it is not easily metabolized in pollen (Hong-Qi and Croes, 1982, Acta Bot. Neerl. 31:113-119). PEG was found to promote tube growth considerably more than a medium containing sucrose. The effect of gibberelins on in vitro germination of various species of corn was also determined (Pfahler et al., 1982, Acta. Bot. Neerl. 31:105-111). The results from these studies indicate that any stimulating effect that GA.sub.3 has is largely genotype specific. The utility of the following nonionic surfactants, Tween 80 (polyoxyethylene sorbitan monooleate), X-114 (alkyl phenoxy-polyethoxy ethanol), and commercial sticker spreader (alkyl olefin aromatic polymers) in combination with a known germination medium (sucrose, bacto-agar, Ca(NO.sub.3).sub.2 and boric acid was studied (Pfahler et al., 1980, Can. J. Bot. 58:557-561). It was found that the more nonionic surfactant, X-114, had the greatest effect on germination.
Results from previous studies indicate that lysis of the pollen cell wall occurs immediately when corn pollen grains are incubated in pollen germination medium which does not contain agar (reviewed in Goss, 1968, Bot. Rev. 34:333-358). Ultimately, the bursting of the pollen and the release of DNAses will follow. A new medium containing calcium, boron, lysine, a glutamic acid, and sucrose was disclosed and said to overcome the problems typically encountered with corn pollen lysing and germination (Saini et al., 1986, Maydica XXXI:227-232). Pollen was incubated in the medium for 15-23 minutes and then stored in an oven at 24.degree. C. for varying lengths of time. The best results were obtained with pollen grains which had been stored 12 hours after anthesis. The germination rate was 60% and after germination could be stored for up to 7.5 hours before bursting. Pollen grains which were germinated at the time of anthesis had a germination rate of 75% but burst after a 5 hour storage period.
Dupuis et al. (1987, Plant Physiol. 85:876-878), isolated viable sperm cells from maize pollen grains. The pollen grains were germinated in a solution which induced lysis to release the pollen contents. No pollen tube growth was observed prior to lysis.
Finally, studies have been conducted to determine the effect of salt on the viability of maize pollen (Dhingra et al., 1985, Ann. Bot. 55:415-420 and Dhingra and Varghese, 1986, Ann. Bot. 57:101-104). The viability and germination of maize pollen was found to be adversely affected by relatively high levels of salinity (e.g. 160 mg/1). In addition, an increase in amylase and invertase activities was observed with increasing salinity resulting in a greater proportion of soluble sugars. Such sugars may be used as respiratory substrates, which may ultimately alter the viability of the pollen.